本文選題：肺腺癌　切入點：RNA編輯　出處：《天津醫(yī)科大學》2016年博士論文

【摘要】：目的:1.研究早期肺腺癌患者基因編碼區(qū)的RNA編輯現(xiàn)象。2.研究腺苷脫氨酶1(Adenosine Deaminases that Act on RNA 1,ADAR1)、腺苷脫氨酶2(Adenosine Deaminases that Act on RNA 2,ADAR2)和鳥氨酸脫羧酶(Ornithine Decarboxylase,ODC)在肺腺癌中表達的臨床意義及預后價值。3.研究抗酶蛋白抑制因子1(Antizyme inhibitor 1,AZIN1)的RNA編輯對A549細胞增殖與轉(zhuǎn)移的影響。方法:1.應用微流控芯片多重聚合酶聯(lián)反應與RNA測序(microfluidics-based multiplex PCR and RNA sequencing)檢測早期肺腺癌患者基因編碼區(qū)的RNA編輯現(xiàn)象。應用熒光定量PCR(RT-q PCR)檢測肺腺癌腫瘤組織與癌旁正常組織中ADAR1、ADAR2和ODC的基因表達水平。2.應用免疫組織化學染色技術(shù)檢測ADAR1、ADAR2和ODC蛋白在肺腺癌腫瘤組織與癌旁正常組織中的表達,分析ADAR1與ODC在肺腺癌中表達的相關(guān)性和預后價值。3.構(gòu)建穩(wěn)定表達編輯型AZIN1基因的A549細胞(A549-edt/AZIN1)和穩(wěn)定表達野生型AZIN1基因的A549細胞(A549-wt/AZIN1)。應用XTT實驗和Transwell細胞侵襲實驗,研究AZIN1的RNA編輯對于A549增殖和侵襲的影響,應用免疫印跡法檢測ODC蛋白的表達水平。結(jié)果:1.相對于癌旁正常組織,肺腺癌組織的RNA編輯水平升高。在肺腺癌腫瘤組織編輯水平最高的20個基因中,在基因編碼區(qū)的共涉及14個基因位點,包括:COG3,SRP9,COPA,SRP9,CACNA1D,RICTOR,ZNF669,METTL6,FLNA,AZIN1,C20orf30,PODXL,FLNB,PDZD7。2.ADAR1基因和ODC基因在肺腺癌腫瘤組織中的表達顯著高于癌旁正常組織,P(27)0.05。ADAR2基因在肺腺癌腫瘤組織中的表達與癌旁正常組織無統(tǒng)計學差異,其中P(29)0.05。3.ADAR1蛋白定位于細胞核,呈棕黃色或棕褐色顆粒。60.4%的肺腺癌組織ADAR1高表達,而癌旁正常組織僅20.8%為高表達,兩組差異有統(tǒng)計學意義(P(27)0.05)。ADAR1蛋白的表達與肺腺癌患者的TNM分期和淋巴結(jié)轉(zhuǎn)移情況明顯相關(guān)(P(27)0.05);而與性別、年齡、吸煙史、胸膜侵犯情況、腫瘤最大直徑和分化程度無明顯相關(guān)(P(29)0.05)。4.ADAR2蛋白定位于細胞核,呈棕黃色或棕褐色顆粒。10.4%的肺腺癌組織ADAR2高表達,而癌旁正常組織中8.3%為高表達,兩組差異無統(tǒng)計學意義(P(29)0.05)。5.ODC蛋白主要定位于細胞質(zhì),細胞漿呈棕黃色。56.3%的肺腺癌組織ODC高表達,而癌旁正常組織僅12.5%為高表達,兩組差異有統(tǒng)計學意義(P(27)0.05)。ODC蛋白的表達與肺腺癌患者的TNM分期明顯相關(guān)(P(27)0.05);而與性別、年齡、吸煙史、胸膜侵犯情況、腫瘤最大直徑、分化程度和淋巴結(jié)轉(zhuǎn)移情況無明顯相關(guān)(P(29)0.05)。6.經(jīng)過秩相關(guān)性分析,肺腺癌患者中ADAR1與ODC的表達呈正相關(guān)(r=0.832,P(27)0.01)。生存分析顯示ADAR1與ODC的表達與預后相關(guān)。Cox比例風險回歸模型進行多因素生存分析顯示,ADAR1蛋白表達水平、TNM分期是影響肺腺癌患者預后的獨立因素。7.通過XTT實驗測試A549、A549-edt/AZIN1和A549-wt/AZIN1的增殖能力。第一天,三組細胞增殖未出現(xiàn)明顯的差異。第三天,A549-edt/AZIN1與A549-wt/AZIN1的增殖速度明顯高于A549(P(27)0.05)。第六天,A549-edt/AZIN1的增殖速度明顯高于A549-wt/AZIN1(P(27)0.05)。8.通過Transwell細胞侵襲實驗,比較A549,A549-edt/AZIN1和A549-wt/AZIN1穿透Matrigel Invasion Chamber的侵襲能力,結(jié)果顯示:A549-edt/AZIN1(477±68)穿過小室的細胞數(shù)量明顯多于A549-wt/AZIN1(327±42)(P(27)0.05),即A549-edt/AZIN1的侵襲能力比A549-wt/AZIN1的侵襲能力強。9.通過Western blot檢測A549-edt/AZIN1和A549-wt/AZIN1中ODC的表達量,發(fā)現(xiàn)A549-edt/AZIN1和A549-wt/AZIN1中ODC的蛋白表達量高于A549(P(27)0.05),且ODC在A549-edt/AZIN1的表達量高于A549-wt/AZIN1,差異具有統(tǒng)計學意義,P(27)0.05。結(jié)論:1.相對于癌旁正常組織,肺腺癌組織的RNA編輯水平升高。在肺腺癌腫瘤組織編輯水平最高的20個基因中,在基因編碼區(qū)的共涉及14個基因位點,包括:COG3,SRP9,COPA,SRP9,CACNA1D,RICTOR,ZNF669,METTL6,FLNA,AZIN1,C20orf30,PODXL,FLNB,PDZD7。2.肺腺癌患者腫瘤組織中ADAR1與ODC的表達明顯高于癌旁正常組織,二者在肺腺癌中的表達呈正相關(guān)。肺腺癌患者腫瘤組織與癌旁正常組織中ADAR2的表達無明顯差異,主要為低表達。ADAR1蛋白的表達與肺腺癌患者的TNM分期和淋巴結(jié)轉(zhuǎn)移情況明顯相關(guān)。ODC蛋白的表達與肺腺癌患者的TNM分期明顯相關(guān)。生存分析顯示ADAR1與ODC的表達與預后相關(guān)。Cox比例風險回歸模型進行多因素生存分析顯示,ADAR1蛋白表達水平、TNM分期是影響肺腺癌患者預后的獨立因素。3.編輯型的AZIN1可以促進A549的增殖與轉(zhuǎn)移,同時AZIN1的高編輯狀態(tài)可以上調(diào)ODC蛋白的表達。
[Abstract]:Objective: To study the early 1. lung adenocarcinoma patients and the gene encoding RNA.2. editing of Ada (Adenosine Deaminases that Act 1 on RNA 1, ADAR1 2 (Adenosine), adenosine deaminase Deaminases that Act on RNA 2, ADAR2) and ornithine decarboxylase (Ornithine Decarboxylase, ODC) on the clinical significance and prognostic value of.3. expression in lung adenocarcinoma antizyme inhibitor 1 (Antizyme 1 inhibitor, AZIN1) RNA editing effect on A549 cell proliferation and metastasis. Methods: 1. application of microfluidic chip multiplex polymerase chain reaction and RNA sequencing (microfluidics-based multiplex PCR and RNA sequencing) for early detection of lung adenocarcinoma gene encoding region of RNA edit. Application of fluorescent quantitative PCR (RT-q PCR) ADAR1 detection of cancer and lung cancer adjacent normal tissues, ADAR2 and ODC gene expression of.2. by immunohistochemical staining ADAR1 color detection technology, the expression of ADAR2 and ODC protein in cancer tissues and lung cancer adjacent normal tissues. The expression of ADAR1 and ODC in lung adenocarcinoma correlation and prognostic value of.3. to build a stable expression editing type AZIN1 gene (A549-edt/AZIN1) and A549 cells stably expressing wild-type AZIN1 gene in A549 cells (A549-wt/AZIN1). Application of XTT assay and Transwell invasion assay of AZIN1 cells, RNA editing effects on the proliferation and invasion of A549, the expression level of ODC protein was detected by Western blotting. Results: 1. compared with normal tissues, increased lung adenocarcinoma RNA editing level. In lung adenocarcinoma tumor tissue level editor 20 the highest gene, including in involving a total of 14 loci, the gene encoding COG3, SRP9, COPA, SRP9, CACNA1D, RICTOR, ZNF669, METTL6, FLNA, AZIN1, C20orf30, PODXL, FLNB, PDZD7.2.ADAR1 and ODC gene Gene expression in lung cancer tissues was significantly higher than that in normal tissues, P (27) and no significant difference between the expression of 0.05.ADAR2 gene in human lung adenocarcinoma in tumor tissue and adjacent normal tissues, including P (29) 0.05.3.ADAR1 protein located in the nucleus, brownish yellow or brown granules of.60.4% lung adenocarcinoma high ADAR1 expression, and normal tissues only 20.8% high expression, there was significant difference between two groups (P (27) 0.05) the expression of.ADAR1 protein and lung cancer TNM staging and lymph node metastasis was significantly correlated (P (27) 0.05); and gender, age, smoking history, pleural invasion, was not related to tumor size and degree of differentiation (P (29) 0.05).4.ADAR2 protein located in the nucleus, brownish yellow or brown granules of.10.4% lung adenocarcinoma with high expression of ADAR2, and 8.3% adjacent normal tissues showed high expression, no statistical difference between the two groups The meaning of (P (29) 0.05).5.ODC protein was mainly localized in the cytoplasm, brownish yellow cytoplasm showed high expression of.56.3% in lung adenocarcinoma tissues and normal tissues ODC, only 12.5% high expression, there was significant difference between two groups (P (27) 0.05) the expression of.ODC protein and lung cancer patients. TNM stage was significantly correlated (P (27) 0.05); and gender, age, smoking history, pleural invasion, tumor diameter, was not related to the degree of differentiation and lymph node metastasis (P (29) 0.05) by.6. rank correlation analysis, a positive correlation between the expression of ADAR1 and ODC in patients with lung adenocarcinoma (r=0.832, P (27) 0.01). Survival analysis showed that ADAR1 and ODC.Cox expression and prognosis of regression model for multivariate survival analysis showed that the expression level of ADAR1 protein, TNM.7. stage were independent prognostic factors in patients with lung adenocarcinoma by XTT test A549, A549-edt/AZIN1 and A549-wt/AZIN1 The proliferation ability. The first day, three groups of cell proliferation showed no obvious difference. The third day, A549-edt/AZIN1 and A549-wt/AZIN1 proliferation rate was significantly higher than that of A549 (P (27) 0.05). For sixth days, the proliferation rate of A549-edt/AZIN1 was significantly higher than that of A549-wt/AZIN1 (P (27).8. 0.05) by Transwell invasion assay, A549, A549-edt/AZIN1 Matrigel Invasion and A549-wt/AZIN1 penetrate the invasion ability of Chamber, the results showed that A549-edt/AZIN1 (477 + 68) the number of cells through the cell was higher than A549-wt/AZIN1 (327 + 42) (P (27) 0.05), namely the invasion ability of A549-edt/AZIN1 than the invasion ability of A549-wt/AZIN1 strong.9. expression by Western blot detection of A549-edt/AZIN1 and A549-wt/AZIN1 in the amount of ODC A549-edt/AZIN1 and A549-wt/AZIN1, found in the protein expression of ODC was higher than that of A549 (P (27) 0.05), and the ODC expression of A549-wt/AZIN1 in A549-edt/ is higher than the amount of AZIN1, the difference is Statistical significance, P (27) 0.05. conclusion: 1. compared with normal tissues, increased lung adenocarcinoma RNA editing level. In lung adenocarcinoma tumor tissue editing level 20 genes included in the highest, involving a total of 14 loci, the gene encoding COG3, SRP9, COPA, SRP9. CACNA1D, RICTOR, ZNF669, METTL6, FLNA, AZIN1, C20orf30, PODXL, FLNB, ADAR1 and ODC PDZD7.2. expression in lung cancer tissues was significantly higher than that in normal tissues, two positive expression in lung adenocarcinoma. No significant difference between the expression of ADAR2 in tumor tissues of patients with cancer and lung adenocarcinoma by in normal tissues, mainly for the low expression of.ADAR1 protein expression in lung cancer patients with TNM staging and lymph node metastasis was significantly related to the expression of.ODC protein in lung cancer patients with TNM stage were significantly related. Survival analysis showed that ADAR1 and ODC expression of.Cox and prognosis risk ratio Multivariate analysis of survival showed that ADAR1 protein expression and TNM stage were independent prognostic factors in patients with lung adenocarcinoma..3. editing AZIN1 could promote the proliferation and metastasis of A549. Meanwhile, the high editing status of AZIN1 could upregulate the expression of ODC protein.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2

【相似文獻】

相關(guān)期刊論文 前5條

1 劉樹濤;李慧萍;齊慶遠;;載脂蛋白B RNA編輯機制概述[J];中國農(nóng)學通報;2013年15期

2 張凱;谷氨酸受體通道RNA編輯機制與癲癇[J];國外醫(yī)學.精神病學分冊;2003年01期

3 余文發(fā);趙玉林;王凱;董明敏;;人喉癌Hep-2細胞膜鉀離子通道與RNA編輯酶1的相關(guān)性[J];中國耳鼻咽喉頭頸外科;2008年04期

4 余文發(fā);趙玉林;王凱;董明敏;;RNA編輯酶1 mRNA在喉癌及其癌旁組織的表達及意義[J];臨床耳鼻咽喉頭頸外科雜志;2008年02期

5 ;[J];;年期

相關(guān)博士學位論文 前4條

1 付堯;膠質(zhì)瘤組織中ADAR2剪接異構(gòu)體的表達鑒定及其介導GluA2亞基Q/R位點的RNA編輯[D];吉林大學;2016年

2 黃超;擬南芥葉綠體PPR蛋白AtECB2參與RNA編輯的機理研究[D];華東師范大學;2016年

3 程世釗;RNA編輯在肺腺癌中的臨床意義及作用機制[D];天津醫(yī)科大學;2016年

4 溫硯子;布氏錐蟲假基因來源的siRNA和NATs的鑒定及RNA編輯復合體MRB1亞基功能研究[D];中山大學;2011年

相關(guān)碩士學位論文 前10條

1 劉思妍;野生二粒小麥鹽脅迫相關(guān)miRNA的鑒定及其葉綠體基因RNA編輯位點的預測與分析[D];西北農(nóng)林科技大學;2016年

2 張娟娟;新型非抗生素類篩選標記系統(tǒng)及水稻質(zhì)體RNA編輯的研究[D];華中農(nóng)業(yè)大學;2013年

3 周瀟;柑橘cor8基因的RNA編輯及枳啟動子的低溫誘導驗證[D];湖南農(nóng)業(yè)大學;2015年

4 鄧李坤;小麥葉綠體蛋白質(zhì)編碼基因RNA編輯位點的測定及與返白現(xiàn)象關(guān)系初探[D];陜西師范大學;2012年

5 江媛;棉花葉綠體RNA編輯位點的測定、分析及機制初探[D];陜西師范大學;2011年

6 蔡磊;RNA編輯酶ADAR1對小鼠淋巴細胞增殖能力以及基因表達譜的影響[D];第四軍醫(yī)大學;2007年

7 史彩萍;基于二代測序數(shù)據(jù)研究神經(jīng)母細胞瘤中的RNA編輯現(xiàn)象[D];華東師范大學;2014年

8 霍斌亮;RNA編輯酶ADAR1對小鼠淋巴細胞周期和凋亡的影響[D];第四軍醫(yī)大學;2008年

9 劉海軍;大豆細胞質(zhì)雄性不育系及其保持系線粒體基因RNA編輯研究[D];東北農(nóng)業(yè)大學;2014年

10 王夢醒;粗山羊草葉綠體基因RNA編輯位點的鑒定與分析[D];西北農(nóng)林科技大學;2015年

，

本文編號：1658721